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1. Field of the Invention
The present invention relates to the absolute characterization of microscopic particles in solution. More particularly, the present invention relates to the absolute characterization of microscopic particles, such as polymers and colloids using static light scattering (SLS) and time-dependent static light scattering (TDSLS). In principle, the size range of detectability should run from about 20 Angstroms to 100 microns, with useful measurability in the range from 20 Angstroms to 2 microns, and a preferred range from about 20 Angstroms to 5000 Angstroms. Stated in terms of molar mass, the detectable range of particles should run from about 500 g/mole to 1014 g/mole, with useful measurability in the range of 500 g/mole to 109 g/mole, with a preferred range from about 1000 g/mole to 107 g/mole.
The preferred use of this invention is the determination of average particle masses, static dimensions, interaction coefficients, and other properties, as well as their changes in time, when scattering is from a very large number of particles. This is to be distinguished from turbidometric and nephelometric techniques, in which turbidity or relative scattering of solutions is measured and compared to relative reference solutions, in order to obtain concentrations of particles. The SLS technique employed refers to absolute macromolecular characterization, and not to determinations of concentrations of particulates with respect to specific relative calibrations, etc. This is also to be distinguished from devices which count and characterize single particles, although the present invention can count and characterize single particles, in addition to making SLS measurements. The least number of particles whose scattered light would be detected in the scattering volume (the volume of illuminated sample whose scattering is measured by a given photodetector) would be on the order of 20 and the maximum on the order of 4xc3x971017, with the preferred range being from about 15,000 to 1.5xc3x971013 particles. In terms of concentration of solute (dissolved polymer or colloid) the range would be from about 10xe2x88x928 g/cm3 (for very large particles) to 0.2 g/cm3 (for very small particles) with the preferred range being from about 10xe2x88x926 to 10xe2x88x921 g/cm3. It should be pointed out that SLS in the absolute mode requires optically transparent solutions in which single, not multiple, scattering dominates. Many particle concentration detectors actually work in turbid solutions, which is a different range of conditions entirely.
SLS has proven to be a useful technique not only for characterizing equilibrium properties of microscopic particles, such as molar mass, dimensions and interactions, but also for following time-dependent processes such as polymerization, degradation and aggregation. Measuring the time-independent angular distribution and absolute intensity of scattered light in the equilibrium cases allows the former properties to be determined, according to procedures set forth by Lord Rayleigh, Debye, Zimm and others (e.g. ref. 1). In particular, this invention can be used in conjunction with the well known procedure of Zimm to determine weight average molar mass Mw, z-average mean square radius of gyration  less than S2 greater than z and second virial coefficient A2. Measuring the time-dependent changes in the scattered intensity allows calculation of kinetic rate constants, as well as deduction of kinetic mechanisms and particle structural features (e.g. refs. 2,3). TDSLS can be used to monitor polymerization and degradation reactions, aggregation, gelling and phase separation phenomena (e.g. ref. 4).
In addition to absolute SLS and TDSLS measurements, the present invention can also simultaneously count and characterize individual particles which are much larger than the principal polymer or colloid particles; e.g., the large particles may have a radius of 5 microns, whereas the polymer may have an effective radius of 0.1 micron. The large particles may represent a contaminant or impurity, or may be an integral part of the solution, e.g., bacteria (large particles) produce a desired polymer (e.g., a polysaccharide) in a biotechnology reactor. The number density of bacteria can be followed in time, and the absolute macromolecular characterization of the polysaccharide could also be made (an auxiliary concentration detector would also be necessary if the polysaccharide concentration changes in time).
The present invention involves automatic online mixing and/or dilution of solutions containing polymers and/or colloids in order to provide relative and/or absolute characterization of these microscopic particles in solution. In the following, the term xe2x80x98dilutionxe2x80x99 will be used, because, whenever two or more solutions are mixed, as described herein, the solutes in each will become dilute. The automatic dilution is intended to replace the traditional prior art of manually diluting such polymer/colloid solutions in order to make characterizing measurements, and to extend measurement capabilities to novel situations, especially those involving non-equilibrium (that is, time-dependent) processes, such as polymerization, degradation, aggregation and phase separation. The method can be used in conjunction with a variety of detectors, such as static light scattering (SLS), time-dependent static light scattering (TDSLS), heterogeneous time dependent light scattering (HTDSLS), dynamic light scattering, refractometry, ultraviolet and visible spectrophotometry, turbidometry, nephelometry, viscometry and evaporative light scattering. The automatic, online dilution of polymer and/or colloid solutions will be shown to have broad applicability in many sectors. In referring to the ensemble of SLS, TDSLS and HTDSLS detectors and methods in the following, the term light scattering (LS) will be used for brevity.
In principle, the size range of detectability of the polymers and/or colloids should run from about 20 Angstroms to 100 microns, with useful measurability in the range from 20 Angstroms to 20 microns, and a preferred range from about 20 Angstroms to 5000 Angstroms. Stated in terms of molar mass, the detectable range of particle molar masses should run from about 500 g/mole to 1014 g/mole, with useful measurability in the range of 500 g/mole to 1011 g/mole, with a preferred range from about 1000 g/mole to 1010 g/mole.
This invention focuses on automated methods that are used to characterize equilibrium and non-equilibrium properties of solutions containing polymers and/or colloid particles. Characterization of polymers and colloids via LS detectors is in terms of average particle masses, static dimensions, interaction coefficients, and other properties, as well as their changes in time, when scattering is from a very large number of particles. When large colloidal particles are present, the use of the method in conjunction with HTDSLS also allows the determination of the number density of these particles, information on their dimensions, and, when the system is not in equilibrium, how these properties change in time.
SLS has proven to be a useful technique for characterizing equilibrium properties of microscopic particles, such as molar mass, dimensions and interactions, and TDSLS and HTDSLS for following time-dependent processes such as polymerization, degradation and aggregation. Measuring the time-independent angular distribution and absolute intensity of scattered light in the equilibrium cases allows the former properties to be determined, according to procedures set forth by Lord Rayleigh, Debye, Zimm and others (e.g. ref. 1). In particular, this invention can be used in conjunction with the well known procedure of Zimm to determine weight average molar mass Mw, z-average mean square radius of gyration  less than S2 greater than z and second virial coefficient A2. Measuring the time-dependent changes in the scattered intensity allows calculation of kinetic rate constants, as well as deduction of kinetic mechanisms and particle structural features (e.g. refs. 2,3). TDSLS can be used to monitor polymerization and degradation reactions, aggregation, gelling and phase separation phenomena (e.g. ref. 4).
In addition to absolute SLS and TDSLS measurements, use of the present invention in conjunction with HTDSLS allows simultaneous counting and characterization of individual particles which are much larger than the principal polymer or colloid particles; e.g., the large particles may have a radius of 5 microns, whereas the polymer may have an effective radius of 0.1 micron. The large particles may represent a contaminant or an impurity, or may be an integral part of the solution, e.g., bacteria (large particles) produce a desired polymer (e.g., a polysaccharide) in a biotechnology reactor. The number density of bacteria can be followed in time, and the absolute macromolecular characterization of the polysaccharide could also be made (an auxiliary concentration detector would also be useful if the polysaccharide concentration changes in time).
The method whereby simultaneous, absolute characterization of polymers and number counting of large particles is carried out, is described in U.S. patent application Ser. No. 08/969,386. To optimize the technique, one should make the sample liquid flow relative to the irradiating laser beam (or other light source) in the scattering chamber, so as to produce countable scattering spikes as each large particle passes through the detected portion of the illuminated volume (the xe2x80x98scattering volumexe2x80x99), while ensuring, via correct design of the optical and electronic detection system, that there is on the average less than one large particle in the scattering volume at any given time. This allows the scattering level to recover to the baseline scattering of the pure polymer between the scattering spikes due to the large particles, so that the polymer can be absolutely characterized. The fraction of baseline time termed herein xe2x80x98clear window timexe2x80x99, and is detailed mathematically in ref. 5, wherein the method has recently been demonstrated. In this demonstration, it was first shown that useful characterization of a polymer solution could be made even in the presence of a large amount of particulate contamination. The contaminant was a known amount of 2 micron latex spheres introduced in increasing amounts to an aqueous polymer solution containing the polymer poly(vinyl pyrrolidone), or PVP. Secondly, the ability to simultaneously make absolute characterization of the polymer while the change in time of the large particle population was monitored was demonstrated by monitoring the growth of E. Coli bacteria amidst an aqueous solution of PVP polymer.
2. General Background of the Invention
SLS is currently used for three main purposes in academic, medical and industrial research and development, and industrial quality control; 1) to characterize useful averages of mass, mean square radius of gyration and second virial coefficient for unfractionated particles in equilibrium, using traditional procedures, 2) to characterize heterogeneous populations of particles which have been fractionated by techniques such as size exclusion chromatography and 3) to follow time-dependent processes. As examples of each purpose: 1) A new biological macromolecule or microstructure is isolated, or a new polymer is synthesized, and its average macromolecular characteristics are determined by SLS. A manufacturer of synthetic polymers, for water treatment, paints, coatings, adhesives, etc., would use SLS for quality control of their product. 2) A synthetic or biological polymer sample contains a wide variety of molar masses (polydispersiiy), and it is desirable to determine the mass and dimension distribution using a fractionation technique coupled to SLS. The purpose of this can be for fundamental research into a biological mechanism, to aid development of new products, to establish quality control specifications of new products, or to assess the effects of different chemical or physical treatments on the product, etc. 3) It is desired to determine how quickly a polymer degrades under attack by such agents as enzymes, heat, radiation, ultrasound, etc., and this can be determined by TDSLS. This will guide studies in developing new pharmacological inhibitors or promoters, or resistant plastics, or biodegradable materials. A central problem of great economic interest, in which TDSLS can be used is to have an on-line method for determination of the build up of molecular weight during industrial polymerization processes. A further use of the present invention will be in the simultaneous measurement of SLS and particle counting in heterogenous solutions.
Clearly, SLS and TDSLS have a wide range of applications, including, but by no means limited to products such as pharmaceuticals, foodstuffs, resins, plastics, coatings, inks, adhesives, liposomes, cosmetics, water treatment and paper making chemicals, paints, additives, plasticizers, microencapsulation structures, etc.
Current technology generally consists of a transparent, hollow sample cell, usually of glass or quartz, into which a scattering sample is introduced and through which a light beam (usually from a laser) is passed. The scattered light then passes through the walls of the transparent cell, where photodetectors or fiber optic pick-ups are placed. The signal from the detected scattered light is then processed and the properties of the scattering sample deduced. Such systems require that sample be introduced into the cell, remote from the main sample batch itself. A disadvantage of a transparent sample cell is that it creates interfaces between incident light and the sample which produce unwanted stray light or xe2x80x98glarexe2x80x99. This stray light or glare, constitutes one of the major pitfalls and nuisances in the actual practice of SLS. Such cells are also relatively expensive and require fairly precise alignment for proper performance.
The present invention includes a (preferably miniaturized) submersible probe which can be brought into the sample, rather than vice versa. No transparent cell need intervene between the sample solution, the incident beam and the optical detectors. This reduces the SLS instrument to a small probe, which can be thought of now as a simple lab probe, like that of a pH or conductivity meter, to be used simply and routinely. The probe portion is relatively inexpensive to fabricate, easy to align, and can even be made to be disposable. The photodetectors, signal processing, etc. are normally remote from the probe. It is anticipated that such an SLS/TDSLS probe is substantially more economical, versatile and easy to use than currently available systems. The present invention can be used in a variety of modes for both time-independent and time-dependent measurements; 1) Submersible mode, in which the probe is submerged in a vessel containing sample solution, such as a beaker, test tube, vat, reactor, etc. 2) Fill mode, in which small amounts of sample liquid (about 3 microliters to 30 milliliters, for example) can be simply pipetted, scooped, or otherwise transferred into the probe body. 3) Flow mode, in which by means of an integrally flanged pump, or hydraulic connection to a tube with flowing sample, the sample liquid flows through the invention. This can be used for unfractionated samples, including those undergoing time-dependent processes (polymerization, degradation, cross-linking, etc.), or samples solutions fractionated by SEC or other means. 4) Insert mode in which a standard glass vial or cell containing sample liquid is inserted into the probe body, instead of filling the probe body, immersing it, or flowing the sample through it. This would be used instead of any of the other three modes, when, for example, the sample may be deleterious for the chamber, because of causticity, gelling, precipitation, etc. It can also be convenient and valuable when many samples are independently prepared and are to be measured separately, and/or when the state of prepared samples in sealed cells is to be checked periodically, without disturbing the sample due to flow, pipetting, etc. The demands of a wide variety of users can be satisfied by simply changing low cost, optical probe assembly, since the detection, electronics, computer interfacing and basic software are the same.
The present invention is distinct from other light scattering devices. For example: U.S. Pat. No. 4,616,927 (Phillips, Reece and Wyatt) and U.S. Pat. No. 5,305,073 (Ford) describe the use of highly polished, optically transparent cell for absolute light scattering measurements. The current invention requires no optically transparent cell. Neither ofthese inventions are submersible, nor can either be considered as a xe2x80x98probexe2x80x99 which can go into the sample liquid being measured. Neither, hence, can fulfill the probe function ofthe current invention, and both are also less versatile, and more costly in general. A submersible light scattering probe is presented by U.S. Pat. No. 5,350,922 (Bartz), but is designed for relative measurements of fairly turbid media (e.g. muds in suspension in water). They collect scattered light, indiscriminately, from 0 to 180xc2x0 scattering angles, and hence cannot perform absolute light scattering on samples requiring exact specification of the scattering angle (their system could work for Rayleigh scatterers, i.e. for sizes much smaller than the incident radiation wavelength; since they are chiefly looking at particulates in suspension, which are generally very large, this condition would not be expected to be met). Furthermore, their device does interpose an optically transparent medium between the light source and the sample liquid and the detector and the sample liquid. In addition, that device cannot be used in either xe2x80x98fill modexe2x80x99 nor flow mode, and hence is also considerably less versatile. None of these inventions mentioned have the versatility and interchangeability of the present invention.
The use of simultaneous multi-angle detection is shown in U.S. Pat. No. 3,850,525, xe2x80x9cSimultaneous Multiple Measurements in Laser Photometersxe2x80x9d.
The following patent documents are incorporated herein by reference: U.S. Pat. Nos. 3,850,525; 3,954,342; 4,265,535; 4,363,551; 4,548,500; 4,616,927; 4,995,514; 5,129,723; 5,155,549; 5,235,179; 5,305,073; 5,350,922; 5,434,667; 5,638,174; and Great Britain patent application Ser. No. 2166234. Also incorporated by reference are the following papers: Florenzano, Strelitzki and Reed, Macromolecules, vol.31, pp.7226-7238,1998, xe2x80x9cAbsolute, On-line Monitoring of Molar Mass during Polymerization Reactionsxe2x80x9d; Strelitzki and Reed, Journal of Applied Polymer Science, vol. 73, pp. 2359-2368, 1999, xe2x80x9cAutomated Batch Characterization of Polymer Solutions by Static Light Scattering and Viscometryxe2x80x9d; Schimanowski, Strelitzki, Mullin, and Reed, xe2x80x9cHeterogeneous Time Dependent Static Light Scatteringxe2x80x9d, Macromolecules, (in pressxe2x80x94copy attached).
Online methods of determining polymer and/or colloid properties are becoming increasingly important in both academic and industrial situations. One pressing need in the polymer industry is for automated, online systems that monitor polymerization reactions in bench scale, pilot plant, and full scale reactors. Numerous empirical means are typically used, including viscometric and hydrodynamic sensors, but none provide absolute, online measures of polymer Mw and polydispersity. Because most polymerization reactions are run at high concentration of reacting monomers, and because virtually all physical methods for determining Mw and other intrinsic properties of individual polymers require highly dilute solutions, it is not generally feasible to make such absolute methods directly on the reaction liquid. Rather, dilution must occur. Prior art in making dilutions of polyimer solutions for LS and viscometric characterization, is to achieve this manually, a time-consuming, tedious process, which can only yield data points widely separated in time. Although automatic dilution has been standard practice for many years in instruments such as automatic chemical titrators, which characterize equilibrium properties of solutions, the inventor is unaware of such a practice for polymer/colloid solutions, where totally different detectors need to be used, and a wide array of properties, phenomena and reactions can be monitored online. Ref. 6 details the first use of the online dilution technique in conjunction with LS, viscometric, refractometric and ultra-violet absorption detectors for monitoring polymerization reactions.
Other areas of application for the automated online dilution technique include monitoring degradation and fermentation reactions, stability of solutions, and robotic automation of equilibrium characterization for polymers/colloids.
The main idea behind the current invention is the automatic dilution of a stock of polymer and/or colloid solution so that equilibrium and/or non-equilibrium properties can be determined online. This involves withdrawing a fraction of material from the polymer stock solution vessel, at the same time that other liquid(s) is (are) drawn from another vessel(s). The automatic dilution can take place by a variety of methods. In its simplest form, one or more hydraulic xe2x80x98Txe2x80x99 fittings (or a single multiple-port fitting) with at least two capillaries or tubes can be used. The lengths and internal diameters of the capillaries or tubes can be chosen so as to establish the fraction of material that is automatically and continuously drawn from each vessel. The mixing takes place within the xe2x80x98Txe2x80x99 junction, or that of a multi-port fitting. Other methods include using programmable mixing pumps, set to withdraw specified fractions from two or more vessels, or binary, tertiary, quaternary or more complex pumps, which incorporate the mixing and high pressure outlet pumping capabilities in one unit. Such pumps are traditionally used to form gradients of solvents for use in chromatographic techniques such as high pressure liquid chromatography (HPLC), but the inventor is unaware of their use for the characterization of polymer solutions as set forth herein.
The applications of the automatic dilution technique include, but are not limited to:
1) The monitoring of polymerization reactions (see U.S. patent application Ser. No. 08/969,386), whereby a small quantity of reacting solution is continuously and automatically withdrawn from the reactor, and automatically mixed with one or more solvents, so as to bring the concentration of polymer into a dilute-enough regime so that LS and auxiliary techniques can be used to make an absolute, online characterization of the polymers. When combined with a concentration detector (ultraviolet and/or visible spectrophotometer, refractometer, evaporative light scattering detector, or other) the polymer weight average mass, Mw, and the root mean square radius of gyration, Rg, can be monitored online. In some cases, the degree of monomer conversion will also be monitored online. By adding a flow type viscometer, of either the single or multiple capillary type, the reduced viscosity of the polymer can also be measured online. Sometimes a flow-type viscometer could be used prior to dilution to determine total solution viscosity. Since the reduced viscosity measured is a viscosity average of the entire, normally polydisperse population, it can be combined with the Mw yielded by LS and the concentration detector(s) to give a useful online index of the polydispersity of the polymer as it is produced in the reactor.
2) Automated determination of equilibrium properties of unfractionated polymer solutions can be made. The method allows a single stock solution of polymer to be made and automatically diluted in steps or in a continuous gradient. Coupling of an LS detector then allows determination of Mw, Rg, and the second and third virial coefficients A2 and A3, respectively. If a flow type viscometer is added, the reduced viscosity of the solution can also be determined. This method has been a recently demonstrated in ref. 7 for the characterization of PVP in terms of Mw, Rg, reduced viscosity and A2and A3.
This method can be of considerable utility when 1) A2 and higher virial coefficients are to be determined. Size Exclusion Chromatography (SEC), because it operates at very low polymer concentration, does not permit such determinations. 2) The polymers are too large to be separated by the SEC columns and merely elute in the void volume. 3) The polymers might damage expensive SEC columns, or it is not known which columns can be used to separate the polymers.
Furthermore, the automated technique lends itselfnaturally to robotic automation, which can be of considerable utility in situations where high sample analysis throughput is needed.
3) By combining the automatic dilution technique with HTDSLS online monitoring of bioreactors can be accomplished. In many bioreactors a microbial species, such as a bacterial or yeast population, co-exists and interacts with a polymer population. This includes cases where polysaccharides (e.g. xanthan) are produced by bacteria and traditional fermentation where biopolymers are broken down by yeast or other organisms. Another instance is in paper and pulp processing, where large cellulosic particles are gradually degraded by acids and other agents. Yet another situation arises in polymer reactors, where cross-linked or highly entangled aggregates, as well as spherulites and other polymeric particles may be produced in addition to individual polymer chains. In all these cases, the automatic dilution coupled to HTDSLS and a concentration detector will allow the simultaneous absolute characterization of the polymeric population and number density counting of large particles, and how they evolve in time.
4) The automatic dilution technique can be used to assess the effect of different solvents on a particular polymer, or interacting polymer/polymer or polymer/colloid system online. For example, the inventor and co-workers recently demonstrated how the automatic dilution technique could be used, in conjunction with an online capillary flow viscometer, to study the electroviscous effect in polyelectrolyte solutions (unpublished results, data curves attached in FIG. 3). When a polyelectrolyte solution at a very low initial ionic strength, whose nominal value is that of an added simple electrolyte, is diluted with a stock solution of the same ionic strength, the dilution is actually not isoionic because the counterions of the polyelectrolyte contribute to the initial ionic strength, but are diluted as dilution with the fixed ionic strength stock occurs. Hence the total ionic strength (due to added simple electrolyte and the polyelectrolyte counterions) decreases as this type of dilution proceeds. The consequence is the type of maxima in reduced viscosity vs. polyelectrolyte concentration seen in FIG. 3.
These data represent the first demonstration, to the inventor""s knowledge, of a continuous, online determination of the electroviscous effect. Until now, prior art required manual dilutions, which yielded only a small number of relatively widely separated concentration points.
Many other applications of the automatic dilution technique in the context of testing solvent effects and interactions properties can be envisioned. For example, by using a total of three reservoirsxe2x80x94one with the stock concentration of polyelectrolyte, one with a pure water or low ionic strength solution, and the other with a high ionic strength solutionxe2x80x94the complete ionic strength behavior of a polyelectrolyte at constant concentration could be determined in a single, automated experiment. Coupling LS and viscosity detectors will yield both the change in static dimensions and polymer hydrodynamics in response to the changing solvent composition. Again, until now, state-of-the-art has required tedious, time-consuming manual mixing of solutions to make such studies.
A further field of application is in the area of polymer/polymer, polymer/colloid, and colloid/colloid interactions. Again, using multiple reservoirs, it will be possible to automatically and continuously monitor how polymers and colloids interact as such factors as their concentration, solvent qualities, and additives (e.g. small molecules such as urea) change. This can be of considerable use, for example, in pharmaceutical screening, tests of the flocculating power of new water purification agents, precipitation tests for proteins, and general stability tests for aggregating systems.
Clearly, the online dilution and auxiliary techniques have a wide range of applications, including, but by no means limited to products such as pharmaceuticals, foodstuffs, resins, plastics, coatings, inks, adhesives, liposomes, cosmetics, water treatment and paper making chemicals, paints, additives, plasticizers, microencapsulation structures, etc.
The inventor is unaware of any techniques which use the automatic dilution of polymer and/or colloidal solutions in order to make online characterization of non-equilibrium properties, such as polymerization or degradation. On the other hand, the typical detector train scheme used in conjunction with the invention is quite similar to detector trains used in Size Exclusion Chromatography (SEC).
In SEC a small quantity of a fixed concentration of polymer solution is injected into a column fractionation system, wherein a certain uncontrolled amount of dilution occurs, after which measurements of equilibrium properties are made. Some chromatography techniques also automatically vary the composition of the solvent that a polymer is eluted in. Again, these are for equilibrium determinations, and can never be considered as online means for characterizing reactions and other non-equilibrium processes in polymer solutions.
Attempts at measuring polymerization reactions in real-time are generally performed on the reaction solution, either within the reactor or on sample withdrawn from the reactor. None of these use, to the inventor""s knowledge, the online dilution technique, nor do any use the online dilution technique coupled with LS and concentration detectors, in order to have a continuous, online record of Mw and associated quantities.
Incorporated by reference are the following papers: Florenzano, Strelitzki and Reed, Macromolecules, vol. 31, pp. 7226-7238, 1998, xe2x80x9cAbsolute, On-line Monitoring of Molar Mass during Polymerization Reactionsxe2x80x9d; Strelitzki and Reed, Journal of Applied Polymer Science, vol. 73, pp. 2359-2368, 1999, xe2x80x9cAutomated Batch Characterization of Polymer Solutions by Static Light Scattering and Viscometryxe2x80x9d; Schimanowski, Strelitzki, Mullin, and Reed, xe2x80x9cHeterogeneous Time Dependent Static Light Scatteringxe2x80x9d, Macromolecules, (in pressxe2x80x94copy attached).
The present invention is the first fully submersible SLS probe for absolute macromolecular characterization (as opposed to particle counting, nephelometry, dynamic light scattering, or relative concentration measurements). The optical assembly of the present invention can be completely immersed in the scattering medium. Thus, the present invention includes a scattering probe which can xe2x80x98go intoxe2x80x99 the medium to be measured (e.g. into test tubes, production vats, etc.), and samples of the scattering medium need not be introduced into a transparent sample cell remote from the medium itself, as is done in current systems. In the present invention the probe can be submerged in a variety of harsh environments, as concerns temperature, pressure and solvents, and communicates to the remote electronic and signal processing portion via a harness containing fiber optic cables.
The present invention can be used in several distinct modes (immersion, fill mode, insert mode and flow mode), giving it wide versatility. The probe of the present invention is not constrained to be immersed in order to function. A small quantity of sample can also be placed in the optical assembly compartment for measurement in a xe2x80x98fill modexe2x80x99. A sample in a transparent vial or cell can also be placed in the chamber or ring member for measurement. Also, the probe can be hooked into a flowing stream of sample liquid for use in different applications such as polymer separation (e.g. size exclusion chromatography), and on-line, unfractionated flows of polymers in a vessel in equilibrium, or undergoing polymerization, aggregation, cross-linking or degradation processes.
The present invention can respond to the needs of a wide variety of users and applications by simply changing the inexpensive optical assembly, since the detection, electronics, computer interfacing and basic software are all the same. For example, a miniature probe with a 10 microliter channel could plug into the same xe2x80x98detection/analysisxe2x80x99 back-end as a 50 milliliter optical probe designed for immersion at high temperatures. There is wide room for substitution of different diameter fibers with different acceptance angles, number of photodetectors on the xe2x80x98detection/analysisxe2x80x99 back-end, etc.
The present invention does not require a transparent sample cell for the scattering solution. Unlike all current SLS systems for absolute macromolecular and colloidal characterization, no glass or other transparent cell need intervene between the sample, the detection fibers and the fiber or lens used for introducing the incident beam. Major advantages which this confers includes avoiding the expense, maintenance and cleaning of transparent cells, and minimizing glare and stray light, because the optical assembly is preferably made from a very dark or black material, and hence does not have highly reflective glass and/or other dielectric surfaces causing spurious glare and reflections.
The optical probe portion of the present invention is preferably miniature in scale. Whereas other devices also use only small sample volumes, those devices require that the sample be pumped or injected in through appropriate plumbing. In the present invention, when used in the fill mode, small quantities of sample can be simply pipetted or dropped into the optical assembly compartment, where they reside during the measurement.
The probe can achieve both absolute calibration and self-cleaning simultaneously when immersed in a proper solvent, such as toluene. Furthermore, because of the direct immersion there are no problems with index of refraction corrections associated with cells which do not maintain cylindrical symmetry about an axis perpendicular to the scattering plane. Hence, well-known, non-proprietary standard calibration procedures can be used for each detector.
The versatile scattering chamber is very inexpensive to fabricate and, in some instances, can be even treated as disposable. This contrasts to the generally high cost of the scattering cell/detector assembly in prior art units.
Unlike existing SLS units, the use of fiber optic detectors and narrow beam focusing make the system quite insensitive to alignment. This has the significant advantage of allowing the unit to operate with a simple coarse alignment, whereas a high degree of alignment is normally required in existing systems. This is achieved because the acceptance cone of the fibers is fairly large (typically 9xc2x0) and the beam is collimated to usually less than 100 microns. Hence, at a remove of 3 mm from the fiber, the beam can be moved up and down approximately 0.5 mm for a 9xc2x0 acceptance angle fiber, without significantly changing the amount of scattered light entering the fiber.
Properly minimizing the scattering volume with a focused beam and using fiber optic detectors and fast detection electronics allow unfiltered samples to be measured, even when no flow or other relative motion between sample and detector exists. This is a major advance, considering that SLS in conventional instruments only became reliable after chemical filtration technologies improved considerably.
The present invention includes a submersible device, which measures relative light scattered at various angles from a large number of scattering particles, from which absolute macromolecular and colloidal characterization is made, via well known, non-proprietary calibration procedures and the well known procedures of Zimm and others. The device need not contain an optically transparent cell interposed between the scattering medium and the incident optic delivering the incident beam and the optical fibers used for detection.
The submersible absolute macromolecular characterization device described in the previous paragraph preferably consists of a completely solid or perforated, or striated or otherwise partially open solid piece, a ring member or a cylinder with a channel inside into which sample liquid enters upon immersion. In this device, polarized or unpolarized incident light (provided by a laser or any other source of visible or ultraviolet light) is led into the channel and spatially filtered with any suitable optical elements such as a tubular lens, miniature convex lens, flat window, fiber optic, irises, etc., or any suitable combination. The light so led in can undergo any necessary degree of collimation, including none, in order to make as narrow an incident beam waist in the detected scattering volume as desired. Scattered light detection is preferably achieved by fiber optic strands, or other fiber optic light conduits, which are exposed to scattered light in the channel, either by virtue of being recessed into the walls of the channel, being flush with the walls of the channel, or protruding into the channel. The degree of collimation of incident light and the diameter of the detecting fibers are combined to optimize the detected scattering volume for the particular sample to be measured. The transmitted incident light is preferably xe2x80x98dumpedxe2x80x99 using any standard beam dump arrangement, such as a hole, Rayleigh horn, prism, etc. The channel is preferably black or blackened to reduce glare and stray light from the incident beam. The delivery and detection optical train elements are preferably gathered into a harness leading to the photodetectors, amplifiers and computer external to the light scattering probe.
Instead of the probe mentioned above which can be immersed in sampling liquid, a different probe can be provided, into whose channel, plugged at one end, rather, a small quantity of sample liquid can be transferred (e.g. by pipette, or by scooping) and therein reside while the scattering measurements are made.
Likewise, a third probe having suitable liquid flow connectors need not be immersed in sampling liquid; instead, through its channel the sample liquid can be made to flow for scattering measurements.
The submersible absolute macromolecular characterization device described above can consist of a ring member, not necessarily closed or circular (e.g. rectangular, elliptical, horseshoe, or any other shape capable of holding the light source fixed relative to the detection fibers (or photodetector when detection fibers are not used)) containing the incident beam delivery optics, beam dump and detection fibers, and which can be immersed directly in a sample liquid for scattering measurements. Alternatively, the submersible absolute macromolecular characterization device described above can consist of a ring member, not necessarily closed or circular (e.g. rectangular, elliptical, horseshoe, etc.) which can be placed inside of a chamber in a cell of appropriate dimension, so as to protect it from the liquid it is immersed in, ambient light or other factors, or to otherwise control how sample liquid reaches the ring member for scattering measurements.
The present invention includes a method whereby any of the devices described above, with appropriately small scattering volume, can be used to measure sample solutions which may contain significant numbers of large scattering contaminants by using fast enough photodetector response to identify, count and eliminate scattering intensity spikes produced by the contaminants, thereby enabling the recovery of the uniform scattering background due to the population of polymers or colloids in the sample. The sample may be either stationary or flowing to accomplish this. Very roughly, the number density of contaminant particles can be on the order of one per scattering volume, so that very tiny scattering volumes allow for relatively higher concentrations of impurity to be present. The identified spikes can be counted and used to assess the particle density of large particles in a solution, and how this number may change in time, as well as simultaneously determining the absolute uniform scattering from a population of polymer or colloids.
The present invention also includes a method whereby the flow mode of the present invention described herein can be used to measure, in real-time, the increase of the weight average molecular weight of polymers being produced in a solution of chemicals undergoing polymerization reactions. This method preferably includes the on-line dilution of the polymer containing solution to bring it into a concentration range where useful, absolute scattering can be measured. This range is where the quantity 2A2cMw is preferably smaller than 1, but can actually be as much as 10. Such dilution can be achieved by the use of hydraulically pulling polymer solution and pure solvent through an hydraulic xe2x80x98Txe2x80x99 or other mixing chamber via a pump or other flow-causing device. A concentration sensitive detector is preferably installed in the line of fluid flow so as to determine in real-time the actual concentration of polymer in the diluted solution. Such a detector may be a refractive index monitor, ultraviolet or visible spectrophotometer, etc.
The present invention also includes a method whereby any of the devices herein described are used to monitor the changes in time of polymer solutions which are undergoing degradation, polymerization, aggregation, gelling, or phase separation.
The present invention also includes a method whereby any of the devices herein described are used to usefully characterize heterogeneous solutions, containing populations of both polymers or colloids and large particulate scatterers, whether either or both of these changes in time or not.
The present invention comprises a kit including light scattering devices of the type described herein, whereby a wide variety of optical probes (with widely varying dimensions, sample capacities, fiber optic types, numbers of angles) made of different materials to withstand different environments can be connected to the same xe2x80x98back-endxe2x80x99 of detection electronics, signal processing and data analysis. The kit can also include the detection electronics, signal processing and data analysis.
The present invention also includes a submersible light scattering probe for the absolute characterization of polymer and colloid solutions which includes a ring member made of a preferably dark, opaque material, having embedded therein a plurality of optical fibers which can be connected to optical detectors remote from the probe. The ends of the optical fibers are preferably in direct contact with the fluid being tested. Instead of submersing the probe in a fluid, fluid can be caused to flow through the probe, placed in the probe, or placed in a transparent vessel placed in the probe. Individual large scattering particles can also be detected, counted, and characterized at the same time absolute characterization of the polymer or colloid solution is performed.
This method preferably includes the on-line dilution of the polymer-containing solution to bring it into a concentration range where useful, absolute scattering can be measured. This range is where the quantity 2A2cMw is preferably smaller than 1, but can actually be as much as around 10 (or even higher). Such dilution can be achieved by the use of hydraulically pulling polymer solution and pure solvent through an hydraulic xe2x80x98Txe2x80x99 or other mixing chamber via a pump or other flow-causing device. A concentration sensitive detector is preferably installed in the line of fluid flow so as to determine in real-time the actual concentration of polymer in the diluted solution.
Such a detector may be a refractive index monitor, ultraviolet or visible spectrophotometer, etc.
FIG. 16 illustrates the scheme used by the inventor et al. (ref. 6) for the online monitoring of a poly(vinyl pyrrolidone), or PVP, reaction.
The present invention also includes a method whereby heterogeneous solutions, containing populations of both polymers or colloids and large particulate scatterers, can be characterized, whether either or both of these changes in time or not.
FIG. 17 shows a three vessel scheme, wherein one vessel contains the polymer or colloid to be characterized, and two other vessels are used, each of which contains different solvents. For example, the polymermight be electrically charged (i.e. a polyelectrolyte) and be dissolved in pure water in the first vessel, whereas solvent #1 might be pure water, and solvent #2 an aqueous solution containing salt. With such an arrangement it would be possible to maintain a fixed polymer concentration by pulling a fixed fraction from the first vessel, while the total salt concentration that the polyelectrolyte is subjected to is continuously changed from pure to very salty water (e.g. 4 molar NaCl). Since the concentration of polyelectrolyte is fixed, and known, a LS detector alone would furnish online information on how the polyelectrolyte conformations and interactions are changing as the solvent becomes more salty. Adding a viscometer would further indicate how the polyelectrolyte hydrodynamic properties are changing with salt concentration.
Similarly, other types of polymers and/or colloids could be in the first vessel, and solvent #1 could be of one type (e.g. pure water) and solvent #2 could be of another type (e.g. an alcohol or other solvent miscible in water). In this way the effects of changing solvent composition on the polymer and/or colloid could be continuously assessed online. Many other variations are possible, since the second solvent could also contain a polymer and/or colloid which interacts with the first polymer and/or colloid solution. The three vessel arrangement hence allows complete phase diagrams to be obtained online. Another area of use would be to determine under what solvent conditions globular polymers, such as proteins, become denatured into random coils.
Extension to more than three vessels is straightforward and is contemplated by the inventor.